Saturation Recovery Experiments Research Articles

High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast.

High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast. The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals. Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E. coli RNR, a substantial upfield shift of the g(1)-value is observed. The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer. (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A). Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K. At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it. From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1). The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.

Measurement of spin-lattice relaxation times with longitudinal detection

New experimental schemes to measure spin-lattice relaxation times T1 on the basis of inversion-recovery and saturation-recovery experiments with longitudinal detection are introduced. With this approach, paramagnetic species with T1 values as short as 20 ns can be measured. Possibilities to reduce unwanted signals and instrumental artifacts are analyzed. An experiment where the signal is induced directly by the time-dependent M2 magnetization is also proposed. Experimental results for organic radicals and defect centers are presented and compared with data obtained with conventional techniques, and a metal complex at 250 K is analyzed where it is very difficult to get information about relaxation times with established methods because of fast spin-spin relaxation.

Saturation recovery in electron spin resonance with pulsed observing microwave power

By appropriate instrumentation, saturation recovery in electron spin resonance has been observed with pulsed observing microwave power. The observing power was ∼100% amplitude modulated with a low duty cycle, in order to sample the magnetization periodically during its recovery from saturation. This technique has the following advantages in the saturation recovery studies of systems having long spin-lattice relaxation times T1: (i) Advantages associated with capacitor-coupled amplifiers are retained. (ii) Higher relaxation amplitudes could be obtained and at the same time the deviation of the observed relaxation time from true spin-lattice relaxation time T1 could be reduced. In the conventional saturation recovery experiments, higher relaxation amplitudes needed to achieve better signal-to-noise ratio require high observing microwave power, which, however, makes the observed relaxation time smaller than T1. The performance and efficacy of this technique have been evaluated using 2,5 di-tert-butyl 1,4 benzosemiquinone anion in n-butanol.

Radiation damping effects of transverse relaxation time measurements

Radiation damping effects on the determination of transverse relaxation times are discussed. Precise measurements in the presence of radiation damping are suggested. In the strong radiation damping case, the spin-spin relaxation time T 2 could not be measured by the simple spin-echo technique, but can be determined by an exponential fitting of the CPMG echo amplitudes while the effective transverse relaxation time T 2* can be determined by extrapolating the line widths in a saturation-recovery experiment to an infinitely short recovery time.

Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.

Effects of sequence and length on imino proton exchange and base pair opening kinetics in DNA oligonucleotide duplexes.

The base catalysed imino proton exchange in DNA oligonucleotides of different sequences and lengths was studied by 1H-NMR saturation recovery experiments. The self-complementary sequences studied were GCGCGAATTCGCGC (I), CGCGAATTCGCG (II), GCGAATTCGC (III), and CGCGATCGCG (IV). The evaluation of base pair lifetimes was made after correction for the measured 'absence of added catalyst' effect which was found to be characterized by recovery times of 400-500 ms for the AT base pairs and 250-300 ms for the GC base pairs at 15 degrees C. End effects with rapid exchange is noticeable up to 3 base pairs from either end of the duplexes. The inner hexamer cores GAATTC of sequences I-II show similar base pair lifetime patterns, around 30 ms for the innermost AT, 5-10 ms for the outer AT and 20-50 ms for the GC base pairs at 15 degrees C. The shorter sequences III and particularly IV show much shorter lifetimes in their central AT base pairs (11 ms and 1 ms, respectively).

Use of high observing power in electron spin resonance saturation-recovery experiments in spin-labeled membranes

Biomolecular collision rates of 14N-containing nitroxide radical labeled stearic acid with similar 15N-containing species in dimyristoylphosphatidylcholine (DMPC) liposomes have been determined using the saturation-recovery electron spin resonance (ESR) method. It is shown that high microwave observing powers can be used to obtain these rates, thereby increasing the signal-to-noise ratio by about ten times relative to methods previously used. The data are analyzed using the rate-equation approach with inclusion of the observing transition probability. A number of solutions to these equations for other experimental situations and for pulse and continuous wave electron–electron double resonance (ELDOR) are also presented. The bimolecular collision rate of labels at the C16 position was found to be 20% greater than for labels at the C12 position, suggesting that the interaction distance differs at these two positions. Even though the high observing power does not affect the ability to extract bimolecular collision rates, the effective spin-lattice relaxation rates are altered. Plots of these latter rates versus power are linear and the slope can be used to determine the absolute microwave field at the sample for a given input power.

Precision in calculated ϱ, T1 and T2 images as a function of data analysis method

In NMR imaging ϱ, T 1 and T 2 images are usually calculated from a set of partial saturation, saturation recovery or inversion recovery experiments with multiple echoes and multiple repetition times. Several methods can be envisaged to extract parameter images from such a set of source images. These methods to a greater or lesser extent take advantage of the fact that a multiple echo/multiple repetition time experiment provides a set of largely independent T 1 and T 2 measurements. In this study several data analysis methods, including weighted and non-weighted averaging of results of independent T 1 and T 2 measurements, weighted and non-weighted averaging of source images prior to data reduction and simultaneous three-parameter fitting, were compared against another in terms of precision, computational efficiency and robustness. The predicted performance of the examined methods was verified by stochastic simulation experiments.

Application of rate equations to ELDOR and saturation recovery experiments on 14N: 15N spin-label pairs

Rate equations describing the time dependence of population differences of the five allowed transitions in an 14N 15N spin-label pair problem are set up. Included in the formulation are the three Heisenberg exchange rate constants and different nitrogen nuclear spin-lattice relaxation rates, electron spin-lattice relaxation rates, and populations for the 14N and 15N moieties. Using matrix algebra, stationary and time-dependent solutions are obtained in a unified theoretical framework. The calculations apply to stationary and pulse electron-electron double resonance and to saturation-recovery ESR. Particular emphasis is placed on short pulse initial excitation, where the transverse relaxation processes are sufficiently slow that only the population difference of the irradiated transition departs significantly from Boltzmann equilibrium during the excitation.

Integral membrane proteins significantly decrease the molecular motion in lipid bilayers: a deuteron NMR relaxation study of membranes containing myelin proteolipid apoprotein.

The influence of the myelin proteolipid apoprotein on lipid chain order and dynamics was studied by 2H NMR of membranes reconstituted with specifically deuterated dimyristoyl phosphatidylcholines. Quadrupolar echo and saturation recovery experiments were fitted by numerical solution of the stochastic Liouville equation, using a model that includes both inter- and intramolecular motions [Meier, P., Ohmes, E. & Kothe, G. (1986) J. Chem. Phys. 85, 3598-3614]. Combined simulations of both the relaxation times and the quadrupolar echo line shapes as a function of pulse spacing allowed unambiguous assignment of the various motional modes and a consistent interpretation of data from lipids labeled on the C-6, C-13, and C-14 positions of the sn-2 chain. In the fluid phase, the protein has little influence on either the chain order or the population of gauche rotational isomers but strongly retards the chain dynamics. For 1-myristoyl-2-[13-2H2] myristoyl-sn-glycero-3-phosphocholine at 35 degrees C, the correlation time for chain fluctuation increases from 20 nsec to 650 nsec and for chain rotation from 10 nsec to 180 nsec, and the gauche isomer lifetime increases from 0.15 nsec to 1.75 nsec, on going from the lipid alone to a recombinant of protein/lipid ratio 0.073 mol/mol. The results are essentially consistent with spin-label ESR studies on the same system [Brophy, P.J., Horvath, L.I. & Marsh, D. (1984) Biochemistry 23, 860-865], when allowance is made for the different time scales of the two spectroscopies.

Population relaxation of CO( v = 1 ) vibrations in solution phase metal carbonyl complexes

Picosecond infrared saturation-recovery experiments were performed to obtain measurements of the vibrational energy lifetimes ( T 1) of CO( v = 1) vibrations ( v≈ 1920-1985 cm −1) of carbonyl-containing metal complexes in dilute, room-temperature solutions. For relaxation of the F 1u CO-stretching vibration of W(CO) 6 in CCl 4, CHCl 3, n-hexane and benzene, T 1, was found to be T 1 = 800±200, 480±50, 140±15 and 60±6 ps, respectively, while the same mode of Cr(CO) 6, in these solvents gave T 1 = 440±70, 295±30, 145±25 and 59±6 ps. Monocarbonyl complexes with coordinated triphenylphosphine groups (TPP) have shorter CO( v = 1 ) lifetimes. These observations are rationalized in terms of molecular structure, intramolecular bonding, solvent interaction, and energy accepting vibrational modes.

Kinetic Properties of the Two‐Level Systems in Glasses with Account of Tunneling

AbstractThe kinetics of two‐level systems in glasses at low temperatures is studied with account of a distribution of their tunneling parameter values. Integral equations describing the process of spectral diffusion in a glass are obtained by the methods of the continuous time random walk theory. The long‐time asymptotics of the process is a superposition of individual and ‘collective’ relaxations, the latter one is characterized by the single relaxation rate. Manifestation of the results in echo saturation recovery experiments in glasses is discussed.

Theory of Longitudinal Relaxation of an AX Spin System under Double Resonance. I. Density Matrix Formulation of Saturation Recovery

Concrete equations of motion describing longitudinal relaxation of an AX system under irradiation of a selective rf field are worked out on the basis of Bloch's density matrix theory. These equations are composed of thermal relaxation and coherent motion of the stirred spin, which determine dynamical characteristics of the spin system. Explicit equations of motion are obtained also for the magnetizations of another spin observed in the saturation-recovery experiment under double resonance. When the stirring field is not too strong to cause any appreciable splitting of the observed line, the recovery of the observed line can be described in terms of several exponential decay terms together with a pair of oscillating terms corresponding to the coherent nutation.

Intracellular compartmentation detected by 31P-NMR in intact photosynthetic wheat-leaf tissue

High-resolution 31P-NMR spectra of wheat-leaf tissue have been obtained. Local variations in bulk magnetic susceptibility in the tissue make direct observation of chemically shifted resonances difficult. Three methods were found of circumventing this problem: (1) magic-angle rotation; (2) saturation recovery experiments; and (3) infiltration of water into the tissue air spaces. In the dark, signals assigned to extravacuolar orthophosphate (P i) at pH close to 7 and vacuolar P i at pH below 6 were detected in the ratio 1:4. When the tissue was illuminated the extravacuolar P i signal reported a more alkaline environment, consistent with expulsion of protons from the chloroplast stroma if this constitutes the principal extravacuolar P i compartment. When tissue was incubated with mannose, mannose 6-phosphate was produced outside the vacuole. At the same time, the vacuolar P i was depleted, while the extravacuolar P i pool remained unchanged.

Acoustic saturation in a glass at low temperatures

An experimental and theoretical study of nonlinear acoustic propagation in a typical glass, fused silica, is presented, at frequencies near 1 GHz and temperatures below 1 K. The data are interpreted within a general framework of pulse propagation in an inhomogeneously broadened two-level absorber. Numerical solutions are compared with the temperature and pulse-width dependence of the critical saturation intensity, as well as with saturation recovery experiments. The data are described by a linewidth ${T}_{2}^{\ensuremath{-}1}$ of the intrinsic tunneling states that agrees with lifetimes obtained from phonon echo experiments. The distribution of relaxation times ${T}_{1}$ which emerges from the analysis of saturation recovery experiments is also understandable within the tunneling model. An improved estimate for the longitudinal deformation potential is ${\ensuremath{\gamma}}_{L}=2.0$ eV. For $T&lt;0.1$ K, the behavior of the pulse velocity is indicative of a coherent propagating mode, i.e., self-induced transparency.

Kinetics for exchange of imino protons in deoxyribonucleic acid, ribonucleic acid, and hybrid oligonucleotide helices.

The lifetime for opening of individual base pairs in a DNA (dCA5G + dCT5G), and RNA (rCA5G + rCU5G), and a hybrid DNA-RNA (rCA5G + dCT5G) helix have been measured by proton nuclear magnetic resonance. The lifetimes were obtained by saturation recovery experiments performed on the hydrogen-bonding imino protons of the Watson-Crick base pairs. In these oligonucleotide helices the observed relaxation rates were dominated by exchange with water, with the magnetic spin-lattice relaxation time of the imino protons possibly being important only at the lowest temperatures in the DNA helix. It was shown that three interior base pairs in the DNA heptamer dCA5G + dCT5G were in the open-limited region, which means that these imino protons exchange every time the base pair opens. The lifetime of the terminal G X C base pairs in the DNA helix are much shorter than the interior A X T base pairs. The pH dependence of the terminal base pairs indicated that the ends of the helix open and close many times before exchange of the imino protons with water takes place. The temperature dependence of the lifetimes of the interior A X T imino protons in the DNA helix showed that these protons exchange only when the double helix has dissociated into single strands. Thus, these lifetimes measure the rate for dissociation of the double helix. The activation energy for this process was found to be 47 kcal/mol. Comparison of the lifetimes of the interior protons in the DNA, RNA, and hybrid helices showed that the rates of dissociation of the RNA and hybrid helices are very similar at 5 degrees C, whereas the rate for the DNA helix was approximately 1 order of magnitude smaller than that for the other two helices. The reasons for the differences in the kinetics of the three helices are discussed, as are the general dynamics of oligonucleotide helices in solution.

DNA conformation, dynamics, and interactions in solution.

The conformation and dynamics of the d(CGCGAATTCGCG) duplex, its analogs containing mismatched base pairs and helix interruptions, and its complexes with actinomycin and Netropsin, bound separately and simultaneously, have been investigated by nuclear magnetic resonance spectroscopy in aqueous solution. Structural information has been deduced from chemical shift and nuclear Overhauser effect parameters, while the kinetics have been probed from line width and saturation recovery experiments on proton and phosphorus markers at the individual base pair level. These studies lead to an improved understanding of the role of nucleic acid sequence on the structure, flexibility, and conformational interconversions in the duplex state. The nuclear magnetic resonance measurements readily identify helix modification and antibiotic binding sites on the nucleic acid and estimate the extent to which the observed conformational and dynamic perturbations are transmitted to adjacent base pair regions.

A general multistate model for the analysis of hydrogen-exchange kinetics.

AbstractIn proton nmr, the chemical exchange rates of slowly exchanging labile hydrogens (with lifetimes in the range ∼ 10 msec – ∼ 1 sec) of peptides, proteins, and nucleic acids can be measured in H2O by a combination of two separate experiments: (1) the transfer of solvent saturation and (2) saturation‐recovery experiments. When these molecules exist in a dynamic equilibrium among different conformations, the experiments cannot be analyzed in a straightforward manner to derive the intrinsic exchange rates. In the present study we have derived analytical expressions for the above two experiments on a biomolecule under certain limiting conditions: (1) the extreme low‐motility limit, where each of the conformational transitions is much slower than the corresponding hydrogen exchange rate with the solvent; (2) the high‐motility limit (EX2 mechanism), which is the opposite extreme of the previous limit; and (3) the low‐motility limit (EX1 mechanism), which is a mixture of limits (1) and (2), i.e., for some of the conformations, the exchange rate with the solvent is much faster than their conformational transition rates, while for the remaining conformations the reverse situation is realized. The results may be considered as a generalization to an arbitrary number of states of the two‐state model treated by Hvidt. Equations have also been derived that are applicable to the iostope exchange method of measuring very slow exchange rates (with life‐times of the order of minutes and longer) in biomolecules. The saturation recovery experiments performed in H2O on the active pentapeptide fragment of thymopoietin serve to illustrate the high‐motility limit. The theoretical formulation presented in this study can be easily adapted to other double‐resonance techniques and also to situations where the kinetics of an arbitrary system existing in a multistate equilibrium are of interest.

Solution Conformation of Peptides through 1H Nuclear Magnetic Double Resonance Measurement of Amide Hydrogen Exchange Rates: Study of the Active Pentapeptide Fragment of Thymopoietin

The amide hydrogen exchange rates of peptides (and other biomolecules) can be related to their secondary and tertiary structures. The exchange rates in the range ∼0.1 to ∼100 s−1 can be conveniently measured in H2O by a double resonance technique involving a combination of two separate 1H NMR experiments: (1) the transfer of solvent saturation and (2) the selective saturation recovery of the labile hydrogen. When a peptide exists in a dynamic equilibrium among several conformations, the interpretation of the above two experiments in terms of intrinsic exchange rates is not straightforward in general. The effect of a multistate conformational averaging is considered in the interpretation of the above two experiments, and analytical expressions have been presented for a few limiting cases of conformational and chemical equilibria. The amide hydrogen exchange rates of the pentapeptide Arg-Lys-Asp-Val-Tyr, the active fragment of the thymic hormone thymopoietin, have been measured in H2O through the transfer of solvent saturation and saturation recovery experiments. The results suggest that this fragment exists in a highly motile dynamic equilibrium among several conformations. In some of these, the Val4 NH hydrogen is shielded significantly from the solvent.

Radiofrequency pulse sequences which compensate their own imperfections

Radiofrequency pulse sequences are described which have the same overall effect as a single 90 or 180° pulse but which compensate the undesirable effects of resonance offset and spatial inhomogeneity of the radiofrequency field H 1. These “composite” pulses are built up from a small number of conventional pulses which rotate the nuclear magnetization vectors about different axes in the rotating frame, while in the intervals between pulses a limited amount of free precession may be allowed to occur. Insight into the way in which pulse imperfections are compensated is obtained by computer simulation of trajectories of families of nuclear spin “isochromats” representing a distribution of H 1 intensity or resonance offset. Composite 90° pulses are suggested as a method of reducing systematic errors in spin-lattice relaxation times derived from progressive saturation or saturation-recovery experiments, and as the preparation pulse of a spin-locking experiment. A test of the effectiveness of the composite 180° pulse sequence has been made by using it for population inversion in a spin-lattice relaxation measurement where T 1 is derived from the null point in the recovery curve, a technique known to be very sensitive to pulse imperfections.